Mapping Local Nucleotide Flexibility by Selective Acylation of 2‘-Amine Substituted RNA

2‘-Amine substitutions in single-stranded oligoribonucleotides react more rapidly with activated esters than 2‘-amine positions in mismatched or duplex RNA substrates. Reactivity does not reflect static solvent accessibility or electrostatics. We infer that acylation of 2‘-amine substituted RNA is sensitive to local nucleotide flexibility. Selective acylation was used to map the structure and magnesium ion dependent conformational changes in tRNA Asp transcripts containing single 2‘-amine substitutions per transcript. Under denaturing conditions, all 2‘-amine substituted RNA positions show similar reactivity. When tRNA Asp transcripts are refolded under strongly native conditions (10 mM Mg 2+, 100 mM NaCl), positions involved in base pairing and known tertiary interactions, including base triples and loop−loop interactions, are protected from modification. In the absence of magnesium ion the acceptor, T- and anticodon stems form stable helices as judged by their low relative 2‘-amine reactivity. In contrast, the D-stem and most tertiary interactions require greater than 1 mM MgCl 2 for stable folding. These results emphasize an interdependence between formation of the D-stem helix and tertiary structure folding for yeast tRNA Asp transcripts. This chemical approach for mapping local RNA flexibility yields results consistent with prior biophysical and biochemical studies emphasizing its utility for mapping local nucleotide environments on small quantities of RNA molecules of any size.